Cytosine deaminase (CD) is an enzyme that converts cytosine to uracil. The bacterial and fungal versions of this enzyme can also convert 5-fluorocytosine (5FC) to 5-fluorouracil (5FU). However, the human and mouse enzyme does not recognize 5FC as a substrate. Bacterial and fungal CD converts 5FC to 5FU, which is then converted to 5-fluoro-deoxyuridine monophosphate (5FdUMP) in all species. 5FdUMP is an irreversible inhibitor of thymidylate synthase, and the accumulation of 5FdUMP leads to cell death by inhibiting DNA synthesis via deoxythymidine triphosphate (dTTP) deprivation.
Because the human CD gene does not convert 5FC to 5FU, the pro-drug 5FC is only toxic in those human cells that are engineered to express a bacterial or fungal CD gene. This has been used to advantage in treating tumors, and is an example of a “suicide gene” system. The tumors are transformed with a bacterial or fungal CD gene, usually by direct injection, implantation or systemic administration of a vector containing the CD gene. The patient is then treated with 5FC and the toxic effects of 5FU lead to death of the transformed cells that continue to divide.
The suicide gene system has been studied extensively as an approach to treat malignant tumors. One of the advantages of the system is that incorporation of the suicide gene into every tumor cell is not necessary for effective therapy; complete tumor responses have been reported in animals when less than 20% of the cells expressed the suicide gene. This phenomenon is known as the “bystander effect,” and is based on the continued toxicity of the drug to neighboring cells when a particular cell dies and releases its drug load (6).
The suicide gene system requires accurate targeting because gene expression in a normal cell followed by exposure to the pro-drug will kill the cell when it attempts to divide. This problem has been addressed by placing the suicide gene under control of a tissue-specific (or preferentially a tumor-specific) promoter so that the gene will be expressed only in a select population of targeted cells. The alpha-fetoprotein promoter, which is preferentially activated in hepatoma cells, is an example of such an approach (8). Because many promoter sequences are not completely tumor specific, the suicide gene may be also be expressed in some amount of healthy tissue. This is not fatal to efficacy, however, because like most chemotherapy, the premise of the treatment is that actively dividing cells are preferentially targeted by the drug.
Although suicide genes are a promising approach for the specific targeting of tumors, there is room for improvement in most aspects of the system. In particular, an enzyme with increased activity would allow the use of lower doses of 5FC, and avoid the reported immunosuppressive effects of high 5FC doses. The present invention provides one such improvement.